Restriction endonucleases are of great importance to molecular DNA cloning, DNA diagnostics and genetic engineering. There is an ever increasing demand for a larger repertoire of recognition specificities. The restriction endonuclease field is at the point where the engineering of cleavage specificities is approachable. BamHI is a good candidate for engineering new specificities. It has been cloned, overexpressed and crystallized. BamHI binding proficient/cleavage deficient (cat-) variants have been isolated by using an in vivo transcriptional interference selection. The selection demands that only cat- variants can lead to spectinomycin resistance (spR). By inserting the BamHI recognition sequence GGATCC into an antisense promoter, transcription can be regulated. Binding of the cat- BamHI to this 'operator' prevents transcription, thereby relieving the transcriptional interference resulting in spR. The 1.95 Angstrom resolution three dimensional structure of BamHI and the 2.2Angstrom resolution DNA/BamHI cocrystal structure has led to a determination of three amino acid residues within a seven amino acid loop involved in recognition of the inner four base pairs of the GGATCC and an additional loop with two adjacent amino acid residues involved with recognition of the outer two base pairs. Cassette mutagenesis which varies the three DNA recognizing residues of a cat- BamHI variant should yield relatively few mutants (8,000). Among these mutants, those which bind to anti-sense promoter operator sequences where the inner four base pairs differ from the cognate GGATCC may easily be selected. After selection of altered binding variants, cleavage will be restored by reversing the cat- mutation. PROPOSED COMMERCIAL APPLICATION: Potentially can lead to custom designed restriction endonucleases for cleavage at any specific DNA sequence.